Genome-wide analysis and expression profiling of the HD-ZIP gene family in kiwifruit

The homeodomain-leucine zipper (HD-Zip) gene family plays a pivotal role in plant development and stress responses. Nevertheless, a comprehensive characterization of the HD-Zip gene family in kiwifruit has been lacking. In this study, we have systematically identified 70 HD-Zip genes in the Actinidia chinensis (Ac) genome and 55 in the Actinidia eriantha (Ae) genome. These genes have been categorized into four subfamilies (HD-Zip I, II, III, and IV) through rigorous phylogenetic analysis. Analysis of synteny patterns and selection pressures has provided insights into how whole-genome duplication (WGD) or segmental may have contributed to the divergence in gene numbers between these two kiwifruit species, with duplicated gene pairs undergoing purifying selection. Furthermore, our study has unveiled tissue-specific expression patterns among kiwifruit HD-Zip genes, with some genes identified as key regulators of kiwifruit responses to bacterial canker disease and postharvest processes. These findings not only offer valuable insights into the evolutionary and functional characteristics of kiwifruit HD-Zips but also shed light on their potential roles in plant growth and development. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10025-7.


Introduction
Transcription factors (TFs) that contain conserved DNA binding domains play a critical role in regulating the expression patterns of target genes by binding to specific cis-elements in the promoter regions of target genes, thereby affecting tissue development and cell differentiation in eukaryotic organisms [1].The homeodomain-leucine zipper (HD-Zip) gene family is a plant-specific class of TFs that have been identified in various plant species, including Arabidopsis thaliana [2,3], rice (Oryza sativus) [4], tomato (Solanum lycopersicum) [5], Brassica rapa [6], maize (Zea mays) [7].The HD-Zip family comprises two conserved domains, the homeodomain and leucine zipper domain [8].The homeodomain (HD) consists of 60 amino acid residues with highly conserved sequences that bind to the target DNA [9], while the leucine zipper (LZ) domain comprises 35-42 amino acid residues and influences the formation of protein dimers [10].Utilizing the sequence characteristics of HD-Zip proteins, the HD-Zip family can be classified into four distinct subfamilies, namely HD-Zip I to IV [8][9][10].In addition to the HD and LZ domains, HD-Zip proteins belonging to different subfamilies also possess other conserved domains, resulting in functional diversification of HD-Zip proteins [2][3][4].
HD-Zip proteins belonging to the HD-Zip II subfamily contain the CPSCE (Cys-Pro-Ser-Cys-Glu) domain and a conserved N-terminal [8].The HD-Zip II subfamily mainly regulates plant responses to light and auxin stimuli [8].AtHB4 and HAT3 can be induced by auxin and are involved in shade-induced growth in Arabidopsis [18].Previous studies have shown that the AtHAT3, AtHB2, and AtHB4 genes co-regulate shoot apical meristem (SAM) formation and cotyledon development in Arabidopsis seedlings [19].The athb4/hat3 double mutant produces severely abaxialized leaves [18].
The HD-Zip III subfamily is characterized by the presence of the steroidogenic acute regulatory protein-related lipid transfer (START) domain, which contains the SAD (START-adjacent domain) and MEKHLA (Met-Glu-Lys-His-Leu-Ala) motifs [8,20].This subfamily mainly plays a role in meristematic formation, lateral organogenesis, polar auxin transport, and vascular system development [20].In Arabidopsis, the HD-Zip III subfamily includes five HD-Zip genes (REV, PHB, PHV, AtHB15, and AtHB8), and all five genes have been shown to directly regulate vascular development [20].Additionally, three genes (REV, PHB, and PHV) also contribute to controlling the abaxial-adaxial patterning of lateral organs [21].
HD-Zip proteins belonging to the HD-Zip IV subfamily contain only the SAD motif and are primarily involved in regulating anthocyanin accumulation, cell differentiation, root development, and trichome formation [8][9][10]18].Previous studies have shown that AtML1 and AtPDF2 play a role in regulating shoot epidermal cell differentiation in Arabidopsis [8,10].Meanwhile, AtGL2 and AtHB10 negatively regulate hair formation to determine trichome and root-hair distribution patterns [22].
The Actinidia genus comprises 54 species and 75 taxa, and is particularly well-known for its most famous fruit, kiwifruit [23].Kiwifruit has become a popular fruit worldwide due to its high vitamin C content and abundant minerals [24,25].Recently, whole-genome de novo sequencing projects and transcriptome sequencing data of A. chinensis (Ac) and A. eriantha (Ae) have been completed, revealing significant variation in flowering time and other vital traits between the two species [26][27][28].The HD-Zip gene family has been shown to play important roles in plant development and stress responses [8][9][10].However, no systematic investigation or functional analysis of the HD-Zip gene family has been reported in kiwifruit.Therefore, in this study, we comprehensively identified the HD-Zip gene family in the genomes of A. chinensis and A. eriantha, and systematically analyzed their gene structures, motif compositions, and chromosomal distributions in both kiwifruit species.
In this study, we aimed to comprehensively analyze the HD-Zip gene family in the A. chinensis and A. eriantha genomes.Our study investigated the gene structure, motif compositions, and chromosomal distributions of the HD-Zip gene family for both species.We also studied the phylogenetic relationships and evolution patterns of the HD-Zip gene family in these two kiwifruit species.In addition, we conducted cis-elements analysis and examined the expression patterns of the HD-Zip genes in various tissues and under different stress conditions.Our findings provide valuable insights into the potential functions of the HD-Zip genes in these two kiwifruit species.

Identification and characterization of HD-Zip proteins in kiwifruit
To identify HD-Zip proteins in kiwifruit, we used the HMMER 3.0 software to search for HD-Zip proteins from the Ac and Ae genomes based on the HD-domain profile (PF00046) and LZ domain profile (PF02183) [27].We found a total of 70 and 55 putative HD-Zips in Ac and Ae, respectively (Fig. 1 and Table S1).We confirmed that all putative HD-Zips in Ac and Ae contained the homeodomain and LZ domain Pfam and CD-search, as well as other conserved domains such as SRPBCC, START, and MEKHLA (Fig. 1).The coding sequence (CDS) length of AcHB genes ranged from 498 to 2535 bp, and the corresponding length of AeHB genes varied from 453 to 2568 bp (Table S1).The predicted length of AcHB proteins ranged from 166 to 845 amino acids, and the corresponding length of AeHB proteins ranged from 151 to 856 amino acids (Tables 1 and 2).The molecular weight range of AcHB proteins was from 19,322.87 to 93,075.14Da, and the range for AeHB proteins was from 17,726.03 to 93,730.17Da (Tables 1 and 2).The theoretical isoelectric point (pI) for AcHB proteins varied from 4.60 to 9.16, and the range for AeHB proteins was from 4.61 to 9.43 (Tables 1 and 2).Most AcHB proteins (63 out of 70) and AeHB proteins (44 out of 55) were predicted to be located in the nucleus (Tables 1 and 2).

Phylogenetic reconstruction of kiwifruit HD-Zips
To investigate the phylogenetic relationships of HD-Zip genes in the two kiwifruit species, we conducted a neighbor-joining (NJ) tree analysis using the fulllength protein sequences of the identified 70 AcHBs, 55 AeHBs, and 48 AtHD-Zips.The results revealed that both AcHBs and AeHBs were classified into four subfamilies (HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV), which was consistent with previous findings in Arabidopsis and other species [28][29][30][31] (Fig. 2).Subfamily I had the most AcHBs (29) and AeHBs (24), while subfamily III had the least AcHBs (9) and AeHBs (7) (Fig. 2, Tables 1 and 2).Subfamily II contained 18 AcHBs and 12 AeHBs, while subfamily IV had 14 AcHBs and 12 AeHBs (Fig. 2, Tables 1 and 2).Both AcHBs and AeHBs grouped with different HD-Zip genes in Arabidopsis, indicating that they probably possessed functional diversifications similar to HD-Zip genes in Arabidopsis and other species (Fig. 2).
The gene structure is an important evolutionary feature of a gene family and provides insights into their function diversification and classification.The number of exons in AcHBs and AeHBs ranged from 1 to 19 (Fig. 4).However, the number of exons in AcHBs and AeHBs belonging to different subfamilies varied greatly (Fig. 4 and Fig. S1).The average number of exons in subfamilies I, II, III, and IV were 3.01, 3.76, 17.43, and 9.65, respectively (Fig. S1).Most AcHB and AeHB genes grouped in the same clade had a similar exon-intron organization (Fig. 4).

Conserved domain analysis and motif composition of kiwifruit HD-Zip
Conserved domains are essential functional elements of proteins, and we identified the conserved domains of kiwifruit HD-Zips to infer their potential functions and functional diversification.Our results showed that the conserved domain architectures of kiwifruit HD-Zips belonging to the same subfamily were more similar than those belonging to different subfamilies (Fig. 5A).In addition to the homeodomain and LZ domain, kiwifruit HD-Zips also harbored several other conserved domains, indicating functional diversification (Fig. 5A).Five AcHBs and three AeHBs belonging to the HD-Zip II subfamily contained the HD-Zip protein N-terminus domain (PF04618) with unknown functions (Fig. 5A).All kiwifruit HD-Zips grouped into the HD-Zip III and IV subfamilies contained the START domain, which was consistent with results in other species (Fig. 5A) [8,20].Moreover, the HD-Zip III subfamily possessed the MEKHLA domain (Fig. 5A).
To infer the potential functions and functional diversification of kiwifruit HD-Zips, we identified their conserved domains and motifs.Conserved domains are typically functional elements of proteins, and our results showed that the conserved domain architectures of kiwifruit HD-Zips belonging to the same subfamily were more similar than those belonging to different subfamilies.In addition to the homeodomain and LZ domain, kiwifruit HD-Zips contained several other conserved domains, indicating their functional diversification.For example, some AcHBs and AeHBs in the HD-Zip II subfamily harbored the HD-Zip protein N-terminus domain with unknown functions, while all kiwifruit HD-Zips in the HD-Zip III and IV subfamilies contained the START domain, consistent with previous findings in other species.We also used MEME software to predict the motif compositions of kiwifruit HD-Zips and identified 12 conserved motifs (Fig. 5B and Fig. S2).The motif numbers of kiwifruit HD-Zips belonging to different subfamilies were significantly different, indicating their different motif organizations (Fig. S3).Almost all kiwifruit HD-Zips contained motif 1-3, which spanned the homeodomain and LZ domain (Fig. 5), while subfamily-specific motifs were also identified.Consistent with the results of the exon-intron structure, kiwifruit HD-Zips showing a closer phylogenetic relationship had more similar conserved motif structures, indicating similar functions.

Synteny analysis of kiwifruit HD-Zips
Gene duplication and loss are key evolutionary forces that contribute to the expansion or contraction of gene families.Duplicated genes can lead to either gene redundancy or new functionalization.To explore the evolutionary history of kiwifruit HD-Zip genes, we conducted synteny analysis between the two kiwifruit species.We visualized the locus relationship of homologous HD-Zip genes and gene duplication events using MCScanX [32].
In the study of Ac, a total of 73 gene duplication events were identified.Similarly, in the analysis of Ae, 34 gene duplication events were discerned (Fig. 6 and Table 3).Interestingly, we found that duplicated gene pairs were randomly distributed across all subfamilies (Fig. 6 and Table 3).Furthermore, all duplicated gene pairs were produced by whole-genome duplication (WGD) or segmental, indicating that WGD or segmental has played a significant role in the expansion of kiwifruit HD-Zips compared to HD-Zips in Arabidopsis thaliana (Table 3).
To investigate the evolutionary forces that drove the expansion or contraction of kiwifruit HD-Zip gene families, we conducted synteny analysis of HD-Zip genes in both kiwifruit species using MCScanX.We identified a total of 73 and 34 gene duplication events in Ac and Ae, respectively (Fig. 6 and Table 3).These events were randomly distributed among all subfamilies and were produced by whole-genome duplication (WGD) or segmental, indicating that WGD or segmental was the primary driver of HD-Zip gene expansion in kiwifruit.To estimate the selection pressure experienced by duplicated genes, we calculated the ratios of nonsynonymous (Ka) versus synonymous (Ks) substitution rates for each duplicated gene pair.The Ka/Ks values ranged from 0.060-0.459and 0.084-0.527for Ac and Ae, respectively (Table 3).All duplicated gene pairs exhibited Ka/Ks values less than one, indicating that the duplicated genes were under purifying selection and that their potential functions were conserved.

Cis-element analysis of promoter regions of kiwifruit HD-Zips
Cis-elements play a crucial role in transcriptional regulation and significantly impact gene function.We extracted the 2000-bp upstream region of each kiwifruit HD-Zip gene and used it to predict the cis-elements.We identified 19 functional cis-elements, including core promoter elements such as TATA-box and CAAT-box, in the promoter regions of kiwifruit HD-Zips.These cis-elements were classified into four subfamilies, including light responsiveness, plant growth and development, hormone-responsive, and stress-responsive subfamily (Figs.S4 and S5).The plant growth and development subfamily was the most abundant within the promoter regions of kiwifruit HD-Zip genes, suggesting that kiwifruit HD-Zips play a significant role in regulating kiwifruit growth and development (Fig. S5).Overall, the number of ciselements in the Ae HD-Zip promoter was lower than that in the Ac HD-Zip promoter (Fig. S5).The cis-element arrangements for the duplicated gene pairs listed in Table 3 were divergently evolved, suggesting specific expression patterns and new functionalization for the duplicated gene pairs (Fig. S4).However, the cis-element arrangements of the orthologous HD-Zip gene pairs for the two species had high similarities, indicating that the orthologous HD-Zip gene pairs possessed similar functions (Fig. S4).

Expression patterns of kiwifruit HD-Zips
To investigate the expression patterns of AcHB genes in different tissues, we collected two transcriptome datasets (Fig. 7A).The first dataset compared the expression profiles of three tissues: leaf, immature fruit, and ripe fruit.
The expression patterns of HD-Zip genes in Ac could be classified into three groups.The first group included most HD-Zip genes, which exhibited low expression levels in Fig. 2 Phylogenetic tree of HD-Zip proteins.The full-length HD-Zip protein sequences from Arabidopsis (At, black gene name and circles), A. chinensis (Ac, red gene name and circles), and A. eriantha (Ae, blue gene name, and circles) were aligned using ClustalX 2.0 with default parameters.The unrooted phylogenetic tree was constructed using MEGA X and the Neighbor-Joining method.Subfamily I to IV were highlighted using red, blue, light green, and green sectors, respectively all three tissues.The expression patterns of HD-Zip genes in the second group showed high tissue-specificity.For example, AcHB37 and AcHB47 were highly expressed in kiwifruit leaf, while three genes (AcHB12/31/59) were expressed in immature kiwifruit fruit, and three other genes (AcHB5/22/25) were highly expressed in ripe kiwifruit (Fig. 7A).Four HD-Zip genes (AcHB10/19/41/61) were expressed in all three tissues (Fig. 7A).The second transcriptome dataset investigated the expression profiles of eight tissues and showed that different HD-Zip family members exhibited divergent expression patterns in different tissues (Fig. 7B).Four HD-Zip genes (AcHB10/19/41/61) were expressed in all eight tissues, indicating their essential role in kiwifruit development (Fig. 7B).The tissue-specific expression patterns of HD-Zip genes in Ac illustrate gene function diversification.
To investigate the potential role of the HD-Zip gene family in regulating kiwifruit resistance or tolerance to pathogen invasion, we analyzed three transcriptome datasets (Fig. 8).In the first dataset, we compared the transcriptional responses of the susceptible cultivars 'hongyang' (HY) to the invasion of Pseudomonas syringae pv.actinidiae (Psa) (Fig. 8A).We found that the expression patterns of several HD-Zip genes were significantly altered upon Psa invasion, such as AcHB25/37 (Fig. 8A).In the second dataset, we compared the expression profiles of two kiwifruit materials with different resistance levels to Psa, namely HT (highly resistant) and HY (susceptible) (Fig. 8B).We divided the expression patterns of HD-Zip genes into four clades and found that expression levels of multiple HD-Zip genes were significantly altered with the invasion of Psa, such as AcHB19/61 (Fig. 8B).LG1 LG14 LG2 LG3 LG5 LG6 LG7 LG8 LG9 LG10 LG11 LG13 LG15 LG22 LG16 LG17 LG18 LG23 LG20 LG24 LG25 LG26 LG27 LG28 LG29 We also found that AcHB45 positively regulated kiwifruit resistance/tolerance to Psa, as its expression level was increased in HT but decreased in HY (Fig. 8B).On the other hand, AcHB5/47 had the opposite effect, indicating that they negatively regulated kiwifruit resistance/tolerance to Psa (Fig. 8B).In the third transcriptome dataset, we investigated kiwifruit responses to the infection of Botrytis cinerea (Fig. 8C).Consistent with the results from the first and second dataset, we found that AcHB61 had a high expression level upon Botrytis cinerea infection (Fig. 8C).These results suggest that HD-Zip genes play an important role in regulating kiwifruit responses to pathogen invasion.
We further investigated the potential role of HD-Zips in regulating postharvest processes of kiwifruit using two transcriptome datasets.Previous research has shown that hydrogen sulfide (H 2 S) can delay the maturation of kiwifruit [33], and the first transcriptome profile estimated kiwifruit responses to the H 2 S treatment.We found that the H 2 S treatment changed the expression levels of HD-Zip genes (Fig. 9A).The expression level of AcHB19 was increased after one day of the H 2 S treatment, suggesting that AcHB19 played a role in delaying the maturation of kiwifruit (Fig. 9a).The expression profiles of three HD-Zip genes (AcHB22/25/41) were reduced after one day of the H 2 S treatment, indicating that those HD-Zips accelerated the maturation of kiwifruit (Fig. 9A).Nitric oxide (NO) is an important signal molecule in regulating the ripening of kiwifruit [34], and the second transcriptome data investigated the expression profiles of kiwifruit in response to the NO treatment (Fig. 9B).Similar to the results of the H 2 S treatment, the NO treatment altered the expression levels of HD-Zip genes (Fig. 9B).For example, the expression level of AcHB19 was increased, and the expression profiles of three HD-Zip genes (AcHB22/25/41) were reduced (Fig. 9B).

Suppression of multiple AcHB Genes expression by Psa infection
In order to elucidate and verify the involvement of the AcHB gene family in response to kiwifruit bacterial canker pathogen (Pseudomonas syringae pv.actinidiae, Psa) infection, an inoculation experiment for bacterial canker was conducted.Concurrently, leveraging transcriptomic data (Fig. 9), six differentially expressed genes were selected for validation through fluorescent quantitative  PCR.Among these, three AcHB genes (AcHB37/45/59) exhibited a significant reduction in expression levels at day 14 post Psa infection (p-value < 0.001, Fig. 10), highlighting their role in the kiwifruit's response to Psa infection.These three genes emerge as crucial candidates for subsequent gene functional studies and mechanistic investigations.

Discussion
The HD-Zip gene family is pivotal in modulating various facets of plant growth, development, and stress response mechanisms [1][2][3]5].While the HD-Zip gene family has been recognized in multiple plant species [5,6,[28][29][30][31], a comprehensive genome-wide analysis of this gene family in kiwifruit remains unexplored.In our research, we undertook a comprehensive genome-wide identification of the HD-Zip gene family in two distinct kiwifruit species: Ac and Ae.We conducted a comparative analysis of HD-Zip characteristics between these species and examined the organization of cis-elements within the promoter regions of the identified HD-Zips across both species.Furthermore, we probed the expression patterns of Ac HD-Zips across varied tissues and under stress conditions.We detected 70 and 55 HD-Zip genes in Ac and Ae, respectively (Fig. 1, Tables 1 and 2).These numbers were higher than the number of HD-Zip family members in Arabidopsis (48 members), indicating that the HD-Zip gene family expanded in kiwifruit.Synteny analysis showed that the duplicated HD-Zip gene pairs in both species were all caused by whole-genome duplication (WGD) or segmental events (Fig. 6 and Table 3).Our genomic analyses also confirmed that three ancient WGD or segmental events occurred in both Ac and Ae genomes (Table 3) [23,26].However, there was a

L
LG29LG 00LG Fig. 6 Chromosome distribution and synteny relationship of HD-Zip genes in two kiwifruit species.The green and blue bars indicated chromosomes for Ac and Ae, respectively.The syntenic gene pairs were connected by lines with different colors.A Ac, B Ae, C Ac-Ae significant difference in the number of HD-Zip genes between Ac and Ae, suggesting that the HD-Zip gene family evolved differently in these two species, which is consistent with previous genomic analyses [23,26].We suggest that the differences in the number and distribution of HD-Zip genes in Ac and Ae may be due to translocation, gene retention, and loss patterns after WGD or segmental.All HD-Zip genes in both species were under purifying selection (Table 3), indicating that these genes are important for kiwifruit development and adaptation.Similar to other species, we divided the kiwifruit HD-Zip gene family into four clades (subfamily I to IV) based on phylogenetic analysis (Fig. 2).We further analyzed the expression profiles of AcHDZips in different tissues and under stress treatments, revealing their potential functions in regulating kiwifruit growth, development, and stress responses.
Our study explored the functional diversification of kiwifruit HD-Zips through analysis of conserved motifs, cis-elements, and expression patterns.In addition to the homeodomain and LZ domain, we found several other conserved domains within kiwifruit HD-Zips, indicative of functional diversification (Fig. 5A).We identified subfamily-specific conserved domains, including the START and MEKHLA domains (Fig. 5A).Furthermore, we observed clade-specific or subclade-specific motifs, suggesting a functional differentiation among HD-Zip genes from different clades (Fig. 5B).However, gene structures and conserved motif organizations were largely consistent across most kiwifruit HD-Zips from the same subclade.A cis-element analysis of the promoter regions of these HD-Zips revealed significant variation in cis-element organization within the same subclade (Fig. S4).We propose that the cis-element organization of HD-Zips from the same subclade regulates their functional

Conclusions
In conclusion, our study provides a comprehensive characterization of the homeodomain-leucine zipper (HD-Zip) gene family in kiwifruit.We systematically identified and categorized 70 HD-Zip genes in Actinidia chinensis (Ac) and 55 in Actinidia eriantha (Ae), classifying them into four subfamilies (HD-Zip I, II, III, and IV) through rigorous phylogenetic analysis.Insightful analyses of synteny patterns and selection pressures highlighted the potential contributions of whole-genome duplication (WGD) or segmental events to the divergence in gene numbers between the two kiwifruit species, with duplicated gene pairs undergoing purifying selection.Additionally, our investigation unveiled tissue-specific expression patterns among kiwifruit HD-Zip genes, identifying certain genes as crucial regulators of responses to bacterial canker disease and postharvest processes.These findings not only enhance our understanding of the evolutionary and functional aspects of kiwifruit HD-Zips but also illuminate their roles in plant growth and development.

HD-Zip gene identification in two kiwifruit species
To identify candidate genes of the HD-Zip family in Ac and Ae genomes, we utilized the Hidden Markov Model (HMM) of the HD-domain profile (PF00046) and LZ domain profile (PF02183) through the software HMMER 3.0 [27].We obtained the whole-genome sequences and protein sequences of both kiwifruit species from the Kiwifruit Genome Database (http:// kiwif ruitg enome.org/) and collected all HD-Zip protein sequences of Arabidopsis from the TAIR website (https:// www.arabi dopsis.org/).

Physicochemical properties analysis of kiwifruit HD-Zip
To further analyze the characteristics of the HD-Zip gene family in the two kiwifruit species, we computed their physicochemical properties, including protein length, theoretical isoelectric point (pI), grand average of hydropathicity (GRAVY), and molecular weight (MW).These properties were calculated using the ProtParam tool available on the ExPASy server (http:// web.expasy.org/ protp aram/).Additionally, we predicted the subcellular localization of kiwifruit HD-Zip proteins using the online software CELLO (v2.5, http:// cello.life.nctu.edu.tw/).

Gene structure, motif analysis, and chromosomal distribution of kiwifruit HD-Zip
The genomic and coding sequences of HD-Zip genes in both kiwifruit species (A.chinensis and A. eriantha) were obtained using TBtools.The gene structures were then visualized using the Gene Structure Display Server (GSDS 2.0).To identify the conserved motifs of HD-Zip proteins, the Multiple Expectation Maximization for Motif Elicitation tool (MEME) was used with a maximum of 12 motifs.The genome locations of HD-Zip genes were extracted from the corresponding GFF file using a Perl script, and the chromosomal distributions were illustrated using MapGene2 Chrome (http:// mg2c.iask.in/ mg2c_ v2.0/).

Construction of phylogenetic tree for kiwifruit HD-Zip proteins
We retrieved the full-length protein sequences of HD-Zip genes from Arabidopsis thaliana, A. chinensis, and A. eriantha and performed multiple sequence alignments using ClustalX with default parameters [35].The resulting aligned sequences were used to construct a phylogenetic tree using the neighbor-joining (NJ) method with a bootstrap value of 3000 in MEGA X software [36].

Syntenic analysis and duplication events identification of kiwifruit HD-Zip
To investigate the syntenic relationship and gene duplication of kiwifruit HD-Zip proteins, we retrieved all protein sequences of Ac and Ae and performed BLASTP alignment with an e-value of 1 × 10-10.We then identified syntenic relationships and duplication patterns of kiwifruit HD-Zip using the MCScanX software with default parameters [29].
The synonymous (Ks) and nonsynonymous (Ka) mutation rates of the duplicated HD-Zip gene pairs were computed using TBtools software [30].To produce collinearity blocks across the whole genome, we conducted syntenic analysis of kiwifruit HD-Zip using the MCScanX software with default parameters [29].Finally, we visualized the collinearity gene pairs of kiwifruit HD-Zip using TBtools [30].

Cis-elements analysis for kiwifruit HD-Zip genes
To analyze the cis-element organization of kiwifruit HD-Zip genes, we obtained the 2000-bp promoter sequences upstream of each HD-Zip gene in kiwifruit using the TBtools software based on the genome sequence and GFF file [37].We predicted and collected cis-elements from the PlantCARE database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html/) [38].

Plant material and bacterial strain
The plantlets of Actinidia chinensis cultivar 'Donghong' were selected for the study and the plant materials were sampled from Guangxi Institute of Botany.The bacterial strain used for the infection experiment was Pseudomonas syringae pv.actinidiae (Psa) strain C48, isolated from infected kiwifruit plants and characterized for its pathogenicity.Psa inoculation was performed following the protocol previous reported [31].

RNA extraction and quantitative PCR
RNA was extracted from incubated leaves of each sample at 0 (before injection of bacteria), 2 and 14 DPI following the instructions provided with the HiPure Plant RNA Kits (Magen, Guangzhou, China).RNA quality was monitored on 1% agarose gels.All primers were judiciously designed utilizing the Primer3Plus online software (http:// www.bioin forma tics.nl/ cgi-bin/ prime r3plus/ prime r3plus.cgi) and were commercially synthesized by Sangon Biotech Co., Ltd., Shanghai, China (Table S2).
The cDNA synthesis from the samples was meticulously conducted through the utilization of the One-step gDNA removal and cDNA synthesis supermix kit (TransGen Biotech Co., Ltd., Beijing, China).Subsequently, this cDNA was employed as the foundational material for all subsequent PCR experiments.
Quantitative PCR (qPCR) assays were executed in a total volume of 20 µL, containing 10 µL of Tip Green qPCR SuperMix (TransGen Biotech Co., Ltd.), 0.2 µM of each primer, 1 µL of cDNA diluted 1:5, and 8.2 µL of ddH2O.The thermal cycling regime consisted of an initial denaturation step at 94 °C for 30 s, followed by 40 amplification cycles at 94 °C for 5 s and 60 °C for 30 s. Subsequently, a gradual temperature increase of 0.5 °C every 10 s was performed to enable melting-curve analysis.Each sample was subjected to triplicate amplification, and all PCR reactions were carried out utilizing the LightCycler 480 instrument (Roche, Basel, Switzerland).The ΔΔCt method was meticulously employed for data analysis, with Achn107181 (kiwifruit Actin gene) serving as the reference gene for normalization.

Fig. 3
Fig. 3 Distribution of HD-Zip genes in Ac A and Ae B genomes

Fig. 4 Fig. 5
Fig.4 Exon-intron structures of HD-Zip genes in two kiwifruit species.The left panel indicated the phylogenetic tree containing AcHB and AeHB proteins; the middle panel showed the ranges of four clades; the right panel showed exon-intron structures of kiwifruit HD-Zip genes.The green rectangle shows exons, the yellow rectangle shows UTRs, and the regular line represents introns

Fig. 8 Fig. 9
Fig. 8 Expression profiles of AcHB genes with invasions of different pathogens.A Expression profiles of AcHBs in the susceptible cultivars 'HY' to Psa invasion.DPI, days post-infection.B Expression profiles of AcHBs in two kiwifruit cultivars infected with Psa.HT and HY represented resistant and susceptible cultivars, respectively.The number following the cultivar name showed hours post the Psa invasion (HPI).C Expression profiles of AcHBs in kiwifruit cultivar 'HY' infected with B. cinerea.The number following the cultivar name showed hours post the Psa invasion (HPI)

Fig. 10
Fig. 10 Expression analysis of AcHBs using RT-qPCR at different times with Psa infection.Actin was used as the internal standard for each gene.DPI, days post incubation

Table 1
Conserved domains of HD-Zip genes in kiwifruit predicted by CDD A and SMART B Ac, A. chinensis; Ae, A. eriantha Protein composition and physiochemical characteristics of HD-Zip proteins in Ac

Table 2
Protein composition and physiochemical characteristics of HD-Zip proteins in Ae

Table 3
HD-Zip duplication events identified in kiwifruit

Table 3
(continued) Fig. 7 Expression profiles of AcHB genes in different tissues.The heatmap indicated log2 rate values of the FPKM (fragments per kilobase of exon model per million mapped reads) values of AcHB genes.A Expression profiles of AcHBs in three tissues.B Expression profiles of AcHB in seven tissues